In analytical chemistry, liquid and gas chromatography techniques have become important tools in the identification of chemical sample components. The basic principle underlying all chromatographic techniques is the separation of a sample chemical mixture into individual components by transporting the mixture in a moving fluid through a porous retentive media. The moving fluid is referred to as the mobile phase and the retentive media has been referred to as the stationary phase. One of the differences between liquid and gas chromatography is that the mobile phase is either a liquid or a gas, respectively.
In the analysis of a sample compound using a gas chromatograph, typically, a supply of inert carrier gas (mobile phase) is continually passed as a stream through a heated column containing porous sorptive media (stationary phase). In the past, chromatographic systems have incorporated columns formed as hollow capillary tubes having an inner diameter in the range of few hundred microns. In such systems, a sample of the subject mixture is injected into the mobile phase stream and passed through the capillary column, which column is typically positioned within an oven. As the subject mixture passes through the capillary column, it separates into its various components. Separation is due primarily to differences in the volatility characteristics of each sample component with respect to the temperature in the column. Column temperature is primarily regulated by oven temperature. A detector, positioned at the outlet end of the capillary column, detects each of the separated components as they exit the column.
Column efficiency (typically referred to in terms of theoretical plates) of capillary columns is dependent on both column length and width. A column having a larger inner diameter must be longer than a column having a smaller inner diameter in order to achieve comparable efficiency. As will be appreciated greater column length results in longer analysis times which can effect sensitivity. However, shorter columns with relatively narrower column inner diameters have, until the present invention, been limited in relation to the techniques available for sample injection. Consequently, certain prior gas chromatographic analyses have been a compromise between sensitivity and efficiency in relation to the column and injection technique utilized.
Moreover, it has been found for the analysis of particular compounds that the technique used to inject the sample compound and carrier gas into the column can have an effect on the qualitative and quantitative precision of the chromatographic analysis. So-called split and splitless injection techniques have been developed for the injection of a sample compound. Split injection techniques are used when it is desired to inject only a portion of the sample compound into the column, for example, when a sample containing high volume concentrations such as an undiluted material is to be analyzed. Splitless injection is used when it is desired to inject the whole sample, i.e., for trace analysis. The problems with both split and splitless techniques are irreproducibility and molecular weight discrimination which can occur in the sample vaporization step.
In Schomburg, G., et al., Sampling Techniques In Capillary Gas Chromatography, Journal of Chromatography, 142 (1977), Amsterdam, pps. 87-102 various GC injection or sampling techniques are studied and compared, including split and splitless injection. It is emphasized that technical difficulties involved in GC sampling are increased when quantitative analyses of trace components in complex mixtures are desired. It was observed that for optimal quantitative and qualitative analysis of complex mixtures, split and splitless techniques proved undesirable. It was concluded that through the use of on-column or direct sample introduction, improvements could be attained in both quantitative and qualitative analysis.
An on-column injection technique results in the sample compound being injected directly onto the column. Such direct injection can, however, result in the loss of volatile components which after rapid vaporization could be ejected from the inlet end of the column due to the pressure created by the injection. One solution to the ejection problem was the development of the cool, on-column injection technique.
In Grob, K., et al., On-Column Injection Onto Glass Capillary Columns, Journal of Chromatography, 151 (1978), Amsterdam, pps. 311-320, on-column injection is discussed in relation to precision and accuracy of qualitative analysis. An on-column injection apparatus is described as including a long narrow needle which is inserted into the column a preferred distance such that the needle tip lies approximately 10 mm within the oven. It is suggested that by removing the input septum, the precision of the analysis will be improved. In prior techniques, the input septum served to seal the inlet and the needle was required to pierce the septum in order to access the column. Grob et al. suggests cooling the injector assembly thereby establishing the cool on-column sample injection technique. In cool on-column sample injection the column in the region of the injection is cooled in relation to the boiling point of the sample in question in order to prevent vaporization and subsequent ejection of components from the column. In such an injection technique, any sample which is flushed from the oven back to the injector is deposited in the injector.
As used herein, quantitative precision refers to the relative standard deviation of the percent area count. Qualitative precision refers to the relative standard deviation of the retention time period.
U.S. Pat. No. 4,269,608 - Sisti et al. discloses an inlet system which provides a cooling gas stream for such a cool on-column injection technique. In Sisti the inlet apparatus is shown, in one embodiment, to have a coil which is said to be capable of drawing heat from, i.e. cooling, the inlet by passing a fluid at a suitable temperature therethrough. However, the disclosed design necessitates the use of long slender syringe needles to ensure that the needle tip is well within the oven region for deposit of liquid sample inside the capillary column. This necessity for long needles has inhibited an otherwise well accepted inlet design from ever being easily or robustly automatable.
In sum, it will be understood from the above that for certain analyses narrow capillary columns and cool on-column injection techniques are preferred. Such a combination, however, will require syringes having needles which are narrower than the column and as shown by Grob et al. are of sufficient length to extend at least 10 mm into the oven. Unfortunately, the combination of needle length and small diameter heretofore has resulted in undesirable bending and buckling, causing system failure. Such phenomena can be predicted in relation to the Euler Formula: ##EQU1## where:
P.sub.cr =Critical Load
A=Cross-Sectional Area
L=Effective Length
R=Radius of Gyration
E=Modulus of Elasticity and
for L/R&gt;150.
One attempt to reduce column diameter and yet allow the use of wider diameter needles has been the development and use of so-called butt connectors. A butt connector can be used to join for example a 530 .mu.m diameter pre-column with a 320 .mu.m diameter analytical column. Unfortunately, the use of butt connectors can result in sacrifice of both efficiency, precision and ease of use.
It will be noted that one advantage of split and splitless techniques is that they can be readily automated. The automation of these techniques, particularly split injection, has significantly improved the precision and non-discrimination of chromatographic analysis. U.S. Pat. No. 4,615,226 - DiNuzzo et al. incorporated herein by reference discloses methods and apparatus for such automation. Automation also dramatically decreases the cost of analysis. Such automated injection devices are commercially available, for example, the HP 7673A, manufactured and sold by the Hewlett-Packard Corporation of Palo Alto, Calif.
Cool on-column injection still has unique advantages in relation to thermally labile or reactive compounds, very volatile compounds and/or wide boiling point mixtures. However, to fully utilize cool on-column injection as a technique, it must be robustly and reliably automatable. Current art limits cool on-column automation to capillary columns having inner diameters of 530 .mu. or larger, thereby greatly limiting the efficiency of the technique. Consequently, a need exists for an automated chromatographic apparatus and method which maximizes robustness and performance of cool on-column injections onto columns having inner diameters of less than 530 .mu.. It will be noted that a practical lower limit of cool on-column injection is approximately under 200 .mu.. This limit is dictated by the need for a syringe needle having a presently manufacturable wall thickness to also have an inner diameter large enough to permit liquid transfer into the inlet (approximately 100 .mu. minimum for most samples). A system that can address this 200-530 .mu. column inner diameter range can greatly broaden the applicability of the cool on-column technique.